Anti-tumor Effects Of Alginate Hydrogel Loaded With R837 And OX40 Antibody On Hepatocellular Carcinoma

Purpose:In recent years,immune checkpoint inhibitors represented by PD1/PD-L1 monoclonal antibody and CTLA-4 monoclonal antibody have shown exciting effects in clinical tumor immunotherapy,but about 80%of patients still cannot benefit from them.It is neccerary to find combination therapy or other drugs related to immune checkpoint.OX40 is one of the co-stimulatory molecules that regulate the second signal required for T cell activation.It is mainly expressed on activated T cells.In contrast to PD-1,OX40 and its ligand can promote the survival of T cells and play a positive role of immune regulation.Toll-like receptor ligands such as CpG and R848 have been shown to increase the expression of OX40 receptors on T cells,and have a synergistic anti-tumor immune effect with OX40 antibody.R837 is a TLR7 agonist,which may increase the expression of OX40 receptor and is expected to cooperate with OX40 to exert anti-tumor efficacy.The transport carrier of the drug is also one of the important factors affecting the efficacy of the drug.The hydrogel behaves good biocompatibility and degradability.A variety of hydrogels have been approved for clinical application for their fuctions such as slow drug releasing,reduced dosages,and the ability to targete tumor microenvironment.Alginate hydrogel is widely used for loading tumor-related drugs because of its simple preparation and direct injection.In view of these,we try to make the following two researches:(1)Construction of a alginate hydrogel drug-carrying system loaded with R837 and OX40 antibodies that can be used for subcutaneous injection;(2)To explore the anti-tumor effects of alginate hydrogel drug-carrying system loaded with R837 and OX40 antibodies.Methods:1.TO construct an injectable alginate hydrogel drug delivery system loaded with R837 and aOX40:different concentrations of sodium alginate solution and calcium chloride solution were used to make hydrogels,and then we compare the gelation time of hydrogels,gel size,degradation time,drug loading,encapsulation rate and drug release rate and so on to select the most suitable concentration of alginate hydrogel that can be used for subcutaneous injection as the drug carrier.2.To explore the anti-tumor effect of the injectable alginate hydrogel drug delivery system loaded with R837 and OX40 antibodies:we construct H22 liver cancer subcutaneous tumor model on ICR mice to explore the synergistic anti-tumor effect of R837 and aOX40,then tumor-killing experiments and flow cytometric detection of tumor infiltrating lymphocytes are preliminary to explore its anti-tumor mechanism;at the same time,we compare the difference of anti-tumor effect between using alginate hydrogel loaded with R837 and aOX40 and direct administration.The near-infrared live imaging of mice was used to verify the sustained release effect of the hydrogel drug-loaded under the skin of mice.Results:1.We use soluble and gel-forming sodium alginate solutions of 2mg/mL and 5mg/mL to prepare hydrogels with 10mg/ml,20mg/ml,and 50mg/ml calcium chloride solutions to compare their gelation time,gel size,degradation time,drug loading,encapsulation rate and drug release rate,etc.The results show that although both components can affect the transaction characteristics and drug loading performance of the hydrogel,the concentration of sodium alginate is the main influencing factor of gel size,degradation time,drug loading,encapsulation rate and drug release rate,and the ratio of 5mg/mL is significantly better than 2mg/mL,while the concentration of calcium chloride mainly affects the gelation time.At 5mg/mL sodium alginate,increasing the calcium chloride concentration from 20mg/ml to 50mg/ml does not significantly improve its drug-carrying performance,and the gelation time is shortened,making it difficult to perform a single subcutaneous injection,so 5mg/mL The concentration ratio of ALG+20mg/mlCaC12 is a more suitable choice for injectable alginate hydrogel.2.In the H22 liver cancer subcutaneous tumor model,the combined application of R837 and aOX40,compared with the PBS group or the single administration group,can significantly inhibit tumor growth and improve the survival time of tumorbearing mice;we furtherly use the above concentration ratio to make the hydrogel loading with R837+aOX40,which shows nearly the same anti-tumor effects in the short term compared with the simply combined application of R837 and aOX40.But in the long-term observation after the drug was stopped,the naked drug group showed a continued growth trend,While the tumors in the drug-loaded hydrogel group were still suppressed and some tumor-bearing mice reached CR,indicating that the drugloaded hydrogel sustained-release system enabled mice long-lasting immunity to resist tumor;in terms of mechanism,we preliminarily confirmed that the joint of R837 and aOX40 promoted the tumor-killing ability of mouse spleen lymphocytes.This ability may come from the fact that R837 promotes the expression of OX40 on the surface of CD4+T lymphocytes.At the same time,the near-infrared live imaging of mice confirmed that the alginate gel constructed by us has effective ability for subcutaneous drug to release slowly.Conclusion:In this experiment,the synergistic effect of the combined application of R837 and AOX40 on inhibiting subcutaneous tumor of H22 liver cancer in ICR mice was preliminarily explored,and the alginate saline gel with appropriate concentration and ratio was prepared.Subcutaneous injection of the hydrogel loaded with R837 and AOX40 could further promote the anti-tumor effect of the combined therapy and enable mice lasting immunity.It shows good clinical application potential,and provides some feasible ideas and methods for aOX40 combination therapy and its application.